Biomarkers for cancer diagnosis and treatment prognosis are important in cancer, and biomarkers useful in cancer drug development fall into three distinct categories. Diagnostic biomarkers allow selection of patients that may have an increased potential for clinical response. In many cases, agents aided in use by these markers target some aspect of the biomarker' s structure or function. Examples include estrogen receptor (ER) or erbB2 expression in breast cancer in relation to ER antagonists or trastuzumab, respectively, or mutated epidermal growth factor (EGF) receptors in non-small cell lung cancer in relation to gefitinib, an EGF-receptor antagonist (Dalton and Friend, 2006). Other types of biomarkers are proximate indicators of a drug's “landing” on its target. These can be utilized to define dosing interval or avoid toxicity. Relevant markers of this type would include proteosome activity in surrogate tissues such as leukocytes after exposure to bortezomib, the novel proteosome inhibitor. A final category of biomarkers is useful as a measure of disease burden, without specific mechanistic reference to how the drug acts. Examples would include chorionic gonadotropin in germ cell neoplasms or CA125 in ovarian cancers (Dalton and Friend, 2006; Mackay et al., 2005). Novel treatments for cancer frequently advance to the clinic without biological predictors of value owing to the lack of validated markers at the time the drug has been defined to possess satisfactory empirical activity with likely acceptable toxicity. Hsp90-directed drugs represent one such novel class of anti-cancer agents that lacks useful, validated markers of either potential clinical benefit, or of drug action on its target. The development of such a biomarker would be of value to the development of these agents (Zhang et al., 2006; Ciocca and Calderwood, 2006).
Historically, Hsp90 was one of a family of proteins induced by a variety of cell stresses e.g. heat, but also by nutrient depletion or ambient acidity, for example. Hsp90 can constitute up to 1-2% of a cell's soluble protein. An extensive body of data supports the concept that Hsps serve as multicomponent machines, “chaperones”, to assist in the proper folding of newly synthesized proteins or refolding of proteins damaged by heat or other stresses, termed “clients” (Ciocca and Calderwood, 2005; Kamal et al., 2004). Hsp90 chaperone function is dependent on its ATP-binding site. When ATP is bound, client protein-Hsp90 complexes in the context of additionally bound “co-chaperone” molecules including Hop40, Hsp70, p50 and p23, can form (Ciocca and Calderwood, 2005; Burger, 2006). Following ATP hydrolysis, a properly folded molecule is released from the Hsp90 complex. If the client protein is improperly or incompletely folded, it is subject to ubiquitination, followed by proteosome-mediated degradation (Kamal et al., 2004; Burger, 2006). Over 500 Hsp90 client proteins have been identified including hormone receptors such as estrogen receptor (ER), progesterone receptor (PR), glucocorticoide receptor (GR) (Pratt and Toft, 2003). Of great interest to cancer treatment is that many of the “clients” of Hsp90 that are processed through its folding function include oncogene products (e.g. c-erbB2, bcr-abl, npm-alk, c-raf, v-src), important regulators of cancer cell growth and cell cycle progression (e.g. cdk4, EGF-R, IGF1-R, telomerase), or apoptosis-related signaling (e.g., akt, mutant p53). This biology has increased enthusiasm for defining modulators of Hsp90 function as a strategy to attack multiple Hsp90 clients (Kamal et al., 2004; Burger, 2006; Pratt and Toft, 2003).
Hsp90 itself exists in two major isoforms, Hsp90α (stress-induced, affording cytoprotection) and Hsp90β (constitutively expressed form, accompanying cellular transformation). Biochemical and functional differences as well as differences in the expression mechanisms and induction of the two isoforms are known (FIG. 2) (Daniel et al., 2005; Sreedhar et al., 2004; Picard, 2004). Hsp90α is encoded by 10 exons in a 5.33 kb genomic DNA and Hsp90β by 11 exons in a 6.88 kb DNA stretch. The human isoforms are about 85% homologous and share a common N-terminal ATP- and geldanamycin binding site (Sreedhar et al., 2004). Their middle domain is the site of client protein binding, and the C-terminal domain is responsible for binding co-chaperone molecules. The main functional difference appears to be that Hsp90α dimerizes more readily than Hsp90β (Sreedhar et al., 2004). Additional Hsp90 analogues are Grp94 (induced, antigen presentation) in the endoplasmic reticulum and TRAP1 (=Hsp75, constitutive active, cell cycle regulation) in the mitochondrial matrix (FIG. 2) (Sreedhar et al., 2004; Picard, 2004). Evidence has emerged that Hsp90α and β are differently expressed in tumors. Hsp90α expression is associated with poor prognosis in breast and pancreas carcinoma, whereas Hsp90β expression has been implicated in tumor drug resistance (Ciocca and Calderwood, 2005; Sreedhar et al., 2004). A very recent study showed that Hsp90α but not Hsp90β is found on the cell surface of invasive fibrosarcoma cells and binds to matrix metalloproteinase 2 in a way that is disrupted by geldanamycins (FIG. 2) (Picard, 2004; Eustace et al., 2004). This “translocalization of Hsp90” to the outside of the cell and its resulting potential impact on conformational folding and interaction with proteins of the extracellular micro-environment, open the possibility that Hsp90α and extracellular clients could be used as circulating response markers of geldanamycin treatment.
Hsp90 antagonists and cancer therapy are further related in the invention. The benzoquinoid ansamycins (BA) were originally defined as anti-tumor antibiotics produced by Streptomyces species, which had the unique property of causing reversion of the transformed phenotype of v-src expressing cells. Index members of the class include herbimycin and geldanamycin (DeBoer et al., 1970; Whitesell et al., 1994). Because BAs decreased phosphotyrosine levels in treated cells, they were originally considered “tyrosine kinase antagonists” (Sakagami et al., 1999). However, mechanistic studies revealed that there was little, if any significant activity in causing tyrosine kinase inhibition in purified kinase assays. Moreover, the physical mass of tyrosine kinases decreased in BA-treated cells, leading to the proposal that BAs somehow affected the “intracellular environment” of the tyrosine kinases (Sakagami et al., 1999). To address the basis for these phenomena, Neckers and colleagues at the National Cancer Institute created a solid-phased geldanamycin derivative, and demonstrated selective adsorption of a 90 kd protein that was identified as Hsp90 (Whitesell et al., 1994). Crystallographic studies by Pavletich et al. later confirmed that BAs can bind with high affinity to a pocket on the surface of Hsp90 (Stebbins et al., 1997). Moreover, a basis for tumor cell selectivity of BAs was established with studies performed by Kamal et al. who showed that affinities of BAs to Hsp90 purified from tumors, as opposed to Hsp90 from normal cells, differ (Kamal et al., 2003). The Kd of Hsp90 for geldanamycins was noteworthily lower in tumor cells, and this correlated with an increased BA-sensitive Hsp90 associated ATPase activity in the latter cell types (Kamal et al., 2003). Thus, BAs have emerged as lead structures to perturb the interaction of Hsp90 with client proteins in a way that is correlated with their capacity to inhibit tumor cell growth.
Geldanamycin itself was unsuitable for formulation, and had severe hepatotoxicity in preclinical toxicology studies. Subsequently, 17-allylamino, 17-demethoxygeldanamycin (17AAG), and 17-dimethylaminoethylamino, 17-demethoxygeldanamycin (17 DMAG) were identified by the inventors and others as analogs suitable for formulation and clinical testing (Burger et al., 2004; Smith et al., 2005; Hollingshead et al., 2005). In addition, mechanistic studies showed that 17AAG and 17DMAG functioned as prototypical Hsp90 antagonists in a way exactly analogous to geldanamycin, with activity in preclinical animal models and evidence of anti-angiogenic activity (Burger et al., 2005; Smith et al., 2005; Hollingshead et al., 2005; Kaur et al., 2004; Nimmanapalli et al., 2001; Munster et al., 2001; Solit et al., 2002; Eiseman et al., 2005).
Other classes of Hsp90 antagonists have been discovered in nature (e.g., radicicol) or designed from structural considerations (e.g., EC69, EC97) (Zhang et al., 2006). While a non-geldanamycin chemotype of Hsp90 antagonist would be greatly interesting, none of these other structural classes has yet to reach the clinic, and therefore strategies to define potential markers for Hsp90 modulator effect must at the present time be focused on BAs. However, these strategies will be very useful as clinical trial opportunities are evolved using other chemotypes.
There are currently employed response markers in clinical trials of BAs. While 17DMAG has just entered phase I clinical trials, 17AAG has advanced to phase II clinical trials and has been tested for efficacy in several hundred cancer patients to date. Phase I clinical trial results that include pharmacokinetic and pharmcodynamic endpoint evaluation have been published (Grem et al., 2005; Banerji et al., 2005; Matthew et al., 2005). The pharmacokinetic data showed that drug levels were achieved well in excess of where modulation of Hsp90 associated client proteins is observed in vitro and in in vivo animal model experiments. In the latter studies, the pharmacodynamic activity of the drug was evaluated by measuring Hsp70 activation and/or degradation of cyclin-dependent kinase 4 or Raf-1.
Encouragingly, pharmacodynamic marker studies in a few but statistically insignificant number of cases did reveal that in the tumor cell compartment, 17AAG has been accompanied by changes in Hsp90 client proteins, particularly Raf-1 and CDK4 (Grem et al., 2005; Banerji et al., 2005; Ramanathan et al., 2005; Matthew et al., 2005). Of interest clinically, although no formal responses have been observed, in the solid tumor patients several patients with renal carcinoma and melanoma have had very protracted periods of disease stability, in one noteworthy case of melanoma out to years of treatment (Banerji et al., 2005).
Biomarker studies accomplished by Western blot of samples from either tumor biopsies or peripheral blood mononuclear cells (PBMC) are both laborious and time consuming. In addition, tumor biopsies are only available from a minority of patients, whereas PBMCs represent at best a surrogate for the tumor, and their use for longitudinal studies may be somewhat limited due to the relatively large amount of blood required (Ciocca and Calderwood, 2005).
This led to the recent exploitation of two new biomarkers by Burrows and co-workers, namely insulin-like growth factor binding protein-2 (IGFBP2) and HER-2 extracellular domain that can be readily detected in patient sera by ELISA (Ciocca and Calderwood, 2005). Burrows et al. argue that both proteins are secreted and might be suitable to predict BA response because they are regulated by Hsp90 client proteins and they demonstrate by using a breast cancer xenograft model (BT474) and 5 normal and 20 cancer patient (not treated) sera that IGFBP2 and HER-2 extracellular domain might be valuable pharmacodynamic tools in clinical trials of Hsp90 inhibitors (Ciocca and Calderwood, 2005).
Taking all pharmacodynamic endpoint studies together, neither in animal tumor models nor in patients is there a clear, validated indicator that has sufficient statistical power to predict for sensitivity to BAs. Therefore it would be highly desirable to develop a biomarker of potential responsiveness to BA action, ideally the direct drug target Hsp90, that can be used to predict clinical benefit, or in a functional sense that the drug having had an effect on the tumor's biology at the dose and schedule studied. As recent evidence described that Hsp90α is secreted into the extracellular milieu, an examination of the detectable Hsp90 isoform in patient plasma is informative as to dynamics of Hsp90 modulator action.